Angiogenesis is upregulated in solid tumors, but the microvasculature that forms is more tortuous and permeable than typical vasculature. Traditional cancer therapies have focused on inhibiting angiogenesis to starve tumors. However, more recent evidence suggests that this approach may have deleterious effects because minimizing angiogenesis increases hypoxia in the tumor which is associated with decreased efficacy of chemotherapeutic and radiation treatment. Moreover, incomplete or leaky vessels can facilitate the intravasation of metastatic cells into the vasculature. As such, stabilizing vasculature may be a promising therapeutic approach to minimizing metastasis, increasing chemotherapeutic efficacy and improving drug delivery to the tumor. Significant emphasis has been placed on targeting VEGF, as it is known to play a key role in promoting angiogenesis and causing increased vascular permeability. However, anti-VEGF therapeutics has met with limited success in several cancer types, including metastatic breast cancer. The researchers' exciting, new data indicates that matrix stiffness, mimicking the stiffening that occurs during breast tumor progression, causes increased angiogenic outgrowth and increased endothelial monolayer permeability- notably, these are the same endothelial phenotypes that are attributed primarily to the action of VEGF. Moreover, these data indicate that matrix stiffness augments endothelial permeability response to VEGF, suggesting a crosstalk between VEGF and matrix stiffness-mediated signaling. Given these findings, this project will investigate the hypothesis that matrix stiffening contributes to impaired microvascular integrity in tumors by disrupting endothelial cell-cell adhesion, and correspondingly, inhibition of stiffening and/or endothelial cell response to stiffening can minimize impaired vascular integrity. Here, 3D in vitro models of matrix stiffness, in vivo models of tumor stiffening, advanced in vivo imaging techniques and RNA-seq will be used to investigate the mechanism by which matrix stiffness alters microvascular permeability in the tumor microenvironment. In Aim 1, the synergies between matrix stiffness and VEGF-mediated permeability will be defined. In Aim 2, the effects of mechanical heterogeneities in the matrix on vessel outgrowth and integrity will be investigated. In Aim 3, approaches to inhibit stiffness-induced vascular barrier disruption will be explored. Together, this work will lead to the identification of novel therapeutic targets to normalize tumor vasculature.